1-Aminocyclopropane-1-carboxylate synthase genes from rosa to control ethylene levels in roses

ABSTRACT

A gene which encodes an ACC synthase is isolated from rose plants, specifically Rosa (cardinal red). This gene is modified for expression in transgenic plants. Isolation of high quality mRNA for gene isolation is achieved through use and adaptation of a 2-butoxyethanol precipitation technique using large amount of initial tissue in order to achieve critical mass for precipitation.

This application is a 371 of PCT application PCT/US97/17644 filed onSep. 30, 1997 which is a continuation-in-part of U.S. application Ser.No. 08/724,194 filed on Oct. 1, 1996, now issued as a U.S. Pat. No.5,824,875.

BACKGROUND OF THE INVENTION

This invention relates to the field of compositions and methods forinhibiting the enzyme 1-aminocyclopropane-1-carboxylate (ACC) synthasein rose thereby prolonging the shelf-life of cut flowers as well asreducing leaf yellowing and petal abscission during shipping andstorage.

A variety of factors cause wilting and natural abscission in flowers,particularly after a cutting of the plant or when flowers have beenremoved from the plant. Such factors include increased oxygen levels,wounding, chemical stress, and the plant's own production of ethylene.Of these factors, the plant's production of ethylene, has been shown toplay a key role in natural senescence, the degenerative process whichgenerally leads to controlled cell death in plants, but also in thedegradation of flowers after they have been cut.

Ethylene, in all higher plants, is important to plant growth anddevelopment from seed germination, seedling growth to flowering andsenescence (Abeles, F. B. et al. (1992), In: Ethylene in Plant Biology.Eds. Abeles, F. B. et al., Academic Press, New York, pp 285-291 and1-13; Yang, S. F. et al. (1984), Annu. Rev Plant Physiol:35, 155-189).Ethylene production in plants can also be associated with trauma inducedby mechanical wounding, chemicals, stress (such as produced bytemperature and water amount variations), and by disease. Hormones canalso stimulate ethylene production. Such ethylene, also sometimes called“stress ethylene”, can be an important factor in storage effectivenessfor plants. Moreover, exposure of plant tissue to a small amount ofethylene often may be associated with increased production of ethyleneby other adjacent plants. This autocatalytic effect may be oftenassociated with losses in marketability of plant material during storageand transportation (Abeles et al., supra; Yang et al., supra).

The ethylene biosynthetic pathway in plants was established by Adams andYang (Adams D. O., et al., (1979) Proc. Nat'l Acad Sci USA 76:170-174)). The first step involves the formation ofS-adenosyl-L-methionine (AdoMet) from methionine byS-adenosyl-L-methionine synthetase. AdoMet is then converted into1-aminocyclopropane-1-carboxylate (ACC), the direct precursor ofethylene in higher plants. This conversion is catalyzed by ACC synthase(S-adenosyl-L-methionine methyl thioadenosine-lyase, EC4.4.1.14), therate limiting step in the ethylene biosynthetic pathway. (See alsoKionka C., et al., (1984) Planta 162:226-235; Amrhein N. et al., (1981)Naturwissenschaften 68: 619-620; Hoffman N. E., et al., (1982) BiochemBiophys Res Commun 104:765-770).

Knowledge of the biosynthetic pathway for ethylene formation has beenfundamental in developing strategies for inhibiting ethylene productionin plants. One approach has been to use chemical inhibitors to inhibitthe synthesis or activity of ethylene, two of the most common beingaminoethoxyvinylglycine and aminooxyacetic acid (Rando, R. R., 1974,Science, 185, 320-324 and in Ethylene in Plant Biology, (Abeles, F. B.,et al., eds. Academic Press, p. 285)). However, chemical methods findlimited use because such methods are expensive and the beneficial effectthey provide is generally only short-lived.

A second approach has been to over express ACC deaminase, an enzymewhich metabolizes ACC, thereby eliminating an intermediate in thebiosynthesis of ethylene (Klee, et al., (1991) Cell 3: 1187-1193) (Seealso Theologis, A., et al. (1993), Cellular and Molecular Aspects of thePlant Hormone Ethylene, p. 19-23). Because ACC deaminase is a bacterialenzyme, it is heterologous, and thus, external to the plant. Thus, it isunlikely that this approach will yield a modification that will bestable from generation to generation.

Yet another approach involves attempts to genetically inhibit theproduction of the enzymes involved in the biosynthesis of ethylene or toinhibit the biosynthesis of the enzymes directly. This approach has theadvantage of not only altering the way the plant itself functionsirrespective of external factors but also of presenting a system whichreproduces itself, that is, the altered plant's progeny will have thesame altered properties for generations to come.

Initial efforts to better understand the enzymes which catalyze thereactions in the biosynthesis of ethylene have involved theidentification and characterization of the genes encoding for AdoMetsynthetase, ACC synthase, and ACC oxidase (See also Kende H., 1993, AnnuRev Plant Physiol Mol Biol 44:283-307). Some of the genes encoding forACC synthase have been identified for a number of plants. For instance,ACC synthase sequences have been identified for zucchini (Sato T., etal., (1989) Proc. Natl Acad Sci USA 86:6621-6625), winter squash(Nakajima, N., et al., (1990) Plant Cell Physiol 31:1021-1029), tomato(Van Der Straeten, D., et al., (1990) Proc Natl Acad Sci USA87:4859-4863); (Rottmann, W. H., et al., (1991) J Mol Biol 222:937-961),apple (Dong, J. G., et al., (1991) Planta 185:38-45), mung bean(Botella, J. R., et al., (1992a) Plant Mol Biol 20:425-436; Botella, J.R., et al., (1993) Gene 123: 249-253; Botella, J. R., et al., (1992b)Plant Mol Biol 18: 793-797); Kim, W. T., et al., (1992) Plant Physiol98:465-471), carnation (Park, K. Y., et al., (1992) Plant Mol. Biol.,18, 377-386), Arabidopsis thaliana (Liang, X., et al., (1992) Proc NatlAcad Sci USA 89:11046-11050; Van Der Staeten, D., et al., (1992) ProcNatl Acad Sci USA 89:9969-9973), tobacco (Bailey, B. A., et al., (1992)Plant Physiol 100: 1615-1616), rice (Zarembinski, T. I., et al., (1993)Mol Biol Cell 4: 363-373), mustard (Wen, C. M., et al., (1993) PlantPhysiol 103:1019-1020), orchid (O'Neill, S. D., et al., (1993) PlantCell 5: 419-432), broccoli (Pogson, B. J., et al., (1995) Plant Physiol108:651-657), and potato (Schlagnhaufer, C. D., et al.. (1995) PlantMol. Biol. 28:93-103).

That ACC synthase is involved in the ethylene pathway is confirmed bythe fact that increased levels of ACC synthase mRNA correlate with anincreased activity of ACC synthase in plants during fruit ripening andflower senescence. Similar correlation is also observed in response toexogenous signals caused either by wounding or due to treatment withhormones such as auxin, cytokinin and ethylene. Interestingly, theexpression of different classes of ACC synthase occurs from a variety ofsignals in many plants, e.g. four different ACC synthase genes have beenshown to be differentially expressed in tomato fruit, cell cultures, andhypocotyls during ripening, wounding, and auxin treatment (Olson, D. C.,et al (1991) Proc. Natl. Acad. Sci. USA 88:5340-5344; and Yip, W. K.,(1992) Proc. Natl. Acad. Sci. USA 89:2475-2479). Differential expressionof two ACC synthase genes has also been observed in winter squash duringwounding or by auxin (Nakajima, et al. (1990) Plant Cell Physiol, 31;1021-29 and (1991) Plant Cell Physiol, 32; 1153-63). Similardifferential regulation of expression ACC synthase genes takes place incarnation flowers by wounding or during senescence (Park, K. Y., et al.,(1992) Plant Mol. Biol., 18, 377-386). The evolution of ACC synthasegenes into a multigene family that responds differentially during plantdevelopment or in response to stimuli external to the plant (Rottmann,W. H., et al., (1991) J Mol Biol 222:937-961) may be a reflection of theimportance of ethylene in plants. (See also Slater, A., et al., (1985)Plant Mol Biol 5:137-147). (Smith, C. J. S., et al., (1986) Planta 168;94-100 and Smith, C. J. S., et al. (1988) Nature 334;724-26). (Hamilton,A. J., et al., (1990) Nature 346:284-286; Köck, M., et al., (1991) PlantMol Biol 17:141-142).

The discovery of the foregoing and of other properties has lead to anunderstanding that it may be desirable to attempt to genetically alterthe production of ethylene in plants. This approach, however, may beconsidered in some ways delicate. Elimination of ethylene is not adesired result as in many instances it will kill the plant. Modulationof ethylene—at the appropriate times—is the critical goal, notelimination of it entirely. This has been attempted at least two pointsin the pathway: the production of ACC by ACC synthase, and the oxidationof ACC by a different enzyme, ACC oxidase. Because the ACC synthaseapproach can permit stable modulation and not only total elimination ofethylene, it is a preferred technique. To date, however, successfulreduction of the production of ethylene through an alteration at the ACCsynthase step in the pathway has only been accomplished in one plant,tomato (Oeller, et al. (1991) Science 254:437-39). In spite of theseemingly simple conceptual nature of this goal, the actualaccomplishment of an alteration of the ethylene biosynthetic pathwaythrough the ACC synthase technique has remained elusive. This isparticularly true for the rose plant, perhaps due to the fact that theidentification of full length genes can be difficult for plants. Asdiscussed later, this may, in part, be due to the fact that isolation offull length or high quality RNA has been deemed “notoriously difficult”for plants. (John, M. E., Nucleic Acids Research 20:2381, 1992, andLogemann, J. et al, Anal Biochem 163, 16-20, 1987).

Efforts by others highlight some of the difficulty involved. Recently,Arteca's laboratory (Wang, T. W. et al., (1995) Plant Physiol.109:627-636) studied two cDNA molecules encoding ACC synthase from awhite flower variety of a flowering geranium plant (Pelargonium xhortorum cv Snow Mass Leaves). As their publication explained (perhapsafter the fact), these researchers tried to identify and characterizetwo clones, GAC-1 and GAC-2. In spite of their efforts, they were onlyable to completely identify one of those cDNA gene sequences, GAC-1.Their study examined the expression of these ACC synthase genes indifferent plant parts of the geranium and in response to stress inducedby osmotic changes (sorbitol) or metal ions (CuCl₂). It also evaluatedthe effects of ethylene on auxin 2,4-D induction in geranium leaves. Thestudy indicated that GAC-1 expression was induced only by stress,whereas expression of GAC-2 appeared to be developmentally regulated.Furthermore, these authors speculated about possible future “transfer ofantisense GAC-1, GAC-2 . . . into Pelargonium tissues through theAgrobacterium transformation or particle bombardment.” This confirms adesire in the art for an ACC synthase approach to altering ethyleneproduction in such plants. In spite of this desire, however, theisolation and identification of some, if not all, the ACC synthase genesequences—for geranium remained elusive. In similar fashion, rose aswell has remained elusive.

Although several plant ACC synthase genes have been identified andsequenced, the current invention describes ACC synthase gene sequenceswhich were previously unknown and which are not believed to have beeneasily discoverable. As mentioned, one factor which may have militatedagainst an expectation of successfully cloning a plant gene is theparticular difficulty in obtaining high-quality and full-length RNA fromplants. Indeed, this process has been characterized as “notoriouslydifficult” by at least more than one practitioner of the art (John, M.E., Nucleic Acids Res. 20:2381, 1992 and Logemann, J., et al, AnalBiochem 163, 16-20, 1987)). While this proved to be true for the presentinventor, these difficulties were overcome by assessing a new approachto the RNA isolation process. The current inventor, after findingtraditional RNA isolation methods to be ineffective, was forced todevelop a non-traditional approach described herein. Basically, eventhough those of ordinary skill in the art had long desired to identifysome gene to manipulate to alter the production of ethylene in someplants, in this case, they failed to realize that the problem lay in theneed for a better isolation process. Even though the implementingtechnology for this process had long been available, those in the artapparently failed to realize how to use that technology to achieve theresults now described. To some extent they simply may not have definedthe problem, preventing the achievement the goals sought. Their effortsmay properly be characterized as having taught away from the directiontaken by the present inventor and, thus, the results achieved hereshould be considered unexpected.

Difficulties in isolating full-length mRNA in the specific case ofgeranium and rose are also further reflected by the fact that one of thesequences encoding ACC synthase in a geranium isolated by the currentinventor (clone pPHSacc49), though it may bear some similarity toportions of the clone termed GAC-2 by Wang et al., supra, (which, in anycase, may have been discovered after the making of the presentinvention) is actually considerably longer than GAC-2. This highlightsthe difficulty in successfully isolating a full-length mRNA moleculeusing standard RNA isolation procedures in certain plant materialsincluding roses. However, the high quality RNA (as defined below)isolated by the current inventor is evidenced by the fact that fulllength cDNA clones were obtained in a different plant, and all of themcould be successfully expressed in an in vitro expression system. Ineach case, full length ACC synthase (enzyme) protein is synthesized invitro. In contrast, even later publications by Arteca's group do notdescribe the actual in vitro expression of any of the isolated DNAclones. In fact the cDNA for the GAC-2 gene was never isolated. Rather,only a partial sequence was merely deduced from the sequence of genomicclones.

This is significant because it highlights the difficulty in isolatingand thereby identifying full length ACC synthase genes. Those ofordinary skill in the art had faced the same challenge. Derivation ofDNA encoding ACC synthase from a genomic clone rarely is successful, andtherefore, simply would not necessarily provide a reasonable expectationof success to one of ordinary skill. Only by utilizing a new anddifferent approach did the present invention successfully identify notonly one but several full length ACC synthase gene sequences from thegeranium plant. The same technique applies to the identification of theACC synthase gene sequence from the rose plant. Basically, it was thishigh quality library containing full length cDNA clones which allowedthe present inventor to successfully achieve direct cloning of ACCsynthase cDNA. The prior art did not discover these sequences becausethe genes did not exist in the available libraries. It was this newapproach which overcame the problems faced, but not solved, by othersand resulted in the extraordinary successes described herein. Theextraordinary success of the present invention—a nearly one hundred foldincrease in positive identifications is a consequence of the newtechnique for RNA isolation and cDNA identification, and not the resultof analogous knowledge gained from the efforts of others. Merecomparison to other genes in the same or different plants did not andcould not have yielded the successes described here. The existence ofthe cDNAs of interest in the library was the governing factor. Thus,even with a viable identification process, successful identification ofthe rose ACC synthase gene, let alone the actual alteration of theplants themselves by means of this knowledge, would not have beenlikely.

Additionally, it should be understood that knowledge of the full lengthsequence of a gene from other plants simply does not necessarily leadone to the sequences of the homologous genes in the rose plants. First,as mentioned earlier, the genes encoding ACC synthase have evolved intoa multigene system in some cases. There appears to be no single gene,but rather a family of genes in most cases. Thus, knowledge of one genein one plant species is not certain to lead to one (or several)homologous or analogous genes in another plant species. Second, becauseknown ACC synthase genes are typically so diverse in their nucleotidesequences, knowledge of one would not lead a person of ordinary skill inthe art to an expectation of success in isolating the ACC synthase genefrom rose.

Antisense technology is a well known approach to create a plant thatproduces less of a selected protein. Through this technology, a plant isaltered by introducing a foreign DNA sequence that encodes an mRNAproduct complementary to part or all of the plant's “sense” mRNAencoding the protein. The presence of antisense RNA inhibits RNAfunction within a cell (and whole organism). Antisense RNA can bind in ahighly specific manner to its complementary sense RNA resulting inblockade in processing and/or translation of the sense mRNA. AntisenseRNA may also disrupt interactions between sense mRNA andsequence-specific RNA binding proteins. Antisense technology may beemployed to inhibit the synthesis of an enzyme involved in ethylenebiosynthesis. The genes identified by the current inventor and disclosedherein have been used for the conception and implementation of antisensesequences specific for ACC synthase mRNA. Introduction of DNA encodingsuch antisense RNA sequences into a rose plant is highly probable toresult in a plant which stably produces less ethylene.

The incorporation of antisense RNA in plants as a means to inhibit thesynthesis of enzymes has been described by various investigators.Rothstein, et al. (1987) Proc. Natl. Acad. Sci. U.S.A. 84: 8439, foundthat antisense RNA inhibited nopaline synthase (nos) in tobacco. Smith,C. J. S., et al. (1988) Nature 334: 724, reported that antisense RNAinhibited polygalacturonase in tomato. Others have used antisense RNA toinhibit the synthesis of enzymes involved in ethylene formation. Oeller,P. W., et al., (1991) Science 254: 437-439, expressed RNA antisense toACC synthase in tomato plants. Others have expressed antisense RNA to adifferent ethylene forming enzyme (EFE), ACC oxidase, in carnation andtomato (Michael, M. Z., et al., 1993, In: Pech, J. C., et al., eds.,Cellular and Molecular Aspects of the Plant Hormone Ethylene (KluwerAcademic Publishers, pp. 298-302); Hamilton, A. J., et al. (1990) Nature346: 284-287; Gray, et al. (1993), in Pech, J. C., et al., supra, pp.82-89; Murray, A. J., et al. (1993) in Pech, J. C., et a., supra,, pp.327-328). The above work with antisense RNA may also be applicable toefforts to stably incorporate the sequences identified by the currentinventor and their antisense sequences into a rose plant. Similarly, thesuccess in expressing antisense RNA for ACC synthase in tomato plantsmay also be applicable (Oeller, et al., supra). It is noteworthy, andperhaps surprising, that neither of the foregoing disclosures have ledto the long sought goal of stably altering ethylene production in roseplants. Hence, no altered rose plants expressing reduced levels ofethylene has been described. The incorporation of ACC synthase antisenseDNA into a rose plant has remained elusive because the complete ACC genesequences were not available prior to the present invention. Thediscoveries disclosed herein enable the production of an appropriatelyaltered rose plant which will express ACC synthase antisense sequencesand stably produce reduced levels of ethylene.

SUMMARY OF THE INVENTION

This invention is based on the discovery and cloning of multiple 1-aminocyclopropane-1-carboxylate (ACC) synthase cDNA molecules. In a rose,there is one molecule which represents the ACC synthase gene from Rosa(actually the cardinal red rose cultivar of the rosa genus). Thenucleotide sequence and corresponding amino acid sequence for this geneis disclosed herein. Importantly, this is believed the first report ofthe full-length sequence for this gene, evidenced by the ability of thecDNAs to be expressed in an expression system.

The invention provides a method for genetic modification of rose plantsto control their levels of ethylene. The newly discovered DNA sequences,fragments thereof, or combinations of such sequences or fragments, isintroduced into a plant cell in reverse orientation to inhibitexpression of ACC synthase, thereby reducing the levels of endogenousethylene.

Using the above methods or plant-specific variants of them, transgenicplants are to be developed and monitored for growth and development.Those plants exhibiting prolonged shelf-life with respect to plantgrowth, flowering, and/or reduced yellowing of leaves due to reductionin levels of ethylene are to be selected and propagated as premierproducts with improved properties including reduced leaf yellowing andpetal abscission during shipping and storage.

The present invention is directed to an isolated DNA molecule encodingan ACC synthase enzyme of a rose which DNA molecule hybridizes withpRoseKacc7 (SEQ ID NO: 1) or a functional derivative of the DNA moleculewhich hybridizes with SEQ ID NO: 1.

The isolated DNA molecule is preferably one with substantial sequencehomology with a molecule selected from the molecule set out in SEQ IDNO: 1. In one embodiment, the isolated DNA molecule is that of SEQ IDNO: 1.

In another embodiment, the present invention provides an isolatedprotein encoded by a DNA molecule as described above, or a functionalderivative thereof A preferred protein has an amino acid sequence of SEQID NO: 2 or is a functional derivative thereof.

Also provided herein is an antisense oligonucleotide or polynucleotideencoding an RNA molecule which is complementary to at least a portion ofan RNA transcript of the DNA molecule described above, which RNAmolecule hybridizes with the RNA transcript such that expression of theACC synthase enzyme is altered.

The above antisense oligonucleotide or polynucleotide molecule can befull length or preferably has between six—or ten, twenty, or fifty—and100 nucleotides.

The antisense oligonucleotide or polynucleotide may be complementary toat least a portion of one strand of the nucleotide sequence SEQ ID NO: 1or may be complementary to at least a portion of an RNA sequence encodedby SEQ ID NO: 1. In one embodiment, the antisense oligonucleotide iscomplementary to at least a part of a 5′ non-coding portion of onestrand of the nucleotide sequence SEQ ID NO: 1.

An antisense oligonucleotide as described above may be complementary toat least a part of the nucleotide sequence SEQ ID NO: 1, which part is,for example, from nucleotides 1-50; nucleotides 51-100; nucleotides101-150; nucleotides 151-200; nucleotides 201-250; nucleotides 251-300;301-350; 351-400; 401-450; or 451-500; or any other such contiguousgroup up to nucleotide 500, 1000, or even to the end of the gene.

This invention is her directed to a vector useful for transformation ofa rose plant cell, comprising:

(a) an antisense oligonucleotide or polynucleotide as described above;

(b) regulatory sequences required for expression of the oligonucleotideor polynucleotide in the cell.

The regulatory sequences comprise a promoter active in the cell, whichmay be an inducible promoter or preferably, a constitutive promoter. Thevector preferably further comprise a polyadenylation signal.

In the above vector the promoter is preferably a heterologous promotersuch as a viral promoter. A preferred viral promoter is the CaMV 35Spromoter or a promoter homologous to CaMV35S.

In other embodiments, the promoter is selected from the group consistingof the SSU gene promoter, ribulose bisphosphate carboxylase promoter,chlorophyll a/b binding protein promoter, potato ST-LS1 gene promoter,soybean heat shock protein hsp17.5-E promoter, soybean heat shockprotein hsp17.3-B promoter, phenylalanine ammonia-lyase promoter,petunia 5-enolpyruvylshikimate-3-phosphate synthase gene promoter,Rhizobium meliloti FIXD gene promoter and nopaline synthase promoter.

Also provided is a rose cell transformed with a vector as describedabove, a plantlet or mature rose plant generated from such a cell, or aplant part from such a plant.

The present invention is further directed to a method to alterexpression of an ACC synthase enzyme in a rose cell, plant or a cuttingthereof, comprising:

(a) transforming either a rose cell or a plant with a vector accordingto any of the prior directions; and

(b) allowing the antisense oligonucleotide or polynucleotide to beexpressed and to hybridize with nucleic acid molecules in the cell,plant or cutting which encode the ACC synthase enzyme.

Also provided is a method of producing a rose plant having reducedethylene production compared to an unmodified plant, comprising thesteps of:

(a) transforming a rose plant with a vector as above;

(b) allowing the plant to grow to at least a plantlet stage;

(c) testing the plant for ACC synthase enzymatic activity or ethyleneproduction; and

(d) selecting a plant having altered ACC synthase activity and/oraltered ethylene production compared to an unmodified rose plant.

A rose plant produced as above, or progeny, hybrids, clones or plantparts thereof, preferably exhibits reduced ACC synthase expression andreduced ethylene production.

In another embodiment, the invention is directed to a method forproducing either a rose or a rose variety (or line), characterized byreduced expression or activity of an ACC synthase enzyme and reducedethylene production compared to an unmodified rose or rose variety,comprising producing a rose plant as above and selfing the plant togenerate the variety.

Also provided is a method for producing a variant plant of a non-rosespecies, an ACC synthase gene of which is homologous to a rose ACCsynthase gene, in which variant plant the ACC synthase expression isaltered in comparison to an unmodified plant of the species, comprising:

(a) identifying and isolating an ACC synthase gene of the species byhybridization with a sense DNA molecule as described above;

(b) constructing a vector which comprises an antisense DNA sequenceencoding at least a part of the gene identified in step (a) in anantisense orientation such that

(i) an RNA transcript of the antisense DNA sequence is complementary tothe part of the gene, and

(ii) expression of the antisense DNA sequence alters expression of theACC synthase gene;

(c) transforming a cell of a plant of the species with the vector ofstep (b) to generate a transformed cell; and

(d) regenerating a plant from the transformed cell of step (c), toproduce the variant plant.

The above method is also used to produce a plant variety in a non-roseplant species characterized by reduced expression or activity of an ACCsynthase enzyme and reduced ethylene production compared to aconventional variety of the species, comprising producing a variantplant as above, and selfing the plant to generate the variety.

This invention also provides a method for genetically altering a plant,preferably (but not necessarily) a plant of a low RNA species,comprising the steps of:

(a) isolating mRNA of the plant using the 2-butoxyethanol precipitationtechnique wherein at least about 3-5 grams of plant tissue startingmaterial is used to attain a critical mass amount of RNA forprecipitation;

(b) constructing a cDNA library from the isolated mRNA;

(c) identifying and cloning a desired DNA sequence from the library;

(d) genetically altering the cloned DNA sequence;

(e) transforming cells of the plant or the plant directly with thealtered DNA sequence; and

(f) if done through a cell-based technique, reproducing a plant from thecells which plant expresses the altered DNA sequence,

thereby genetically altering the plant.

In the above method the plant is preferably a species of the genus Rosa.In the above method, the cloned DNA sequence preferably encodes ACCsynthase. The cDNA in the above method is preferably selected from thegroup consisting of SEQ ID NO: 1.

The above method is used to produce a genetically altered rose plant,comprising the steps of:

(a) isolating rose mRNA using a 2-butoxyethanol precipitation techniquewherein at least about 3-5 grams of plant tissue starting material isused to attain a critical mass amount of RNA for precipitation;

(b) constructing a cDNA library from the isolated mRNA;

(c) identifying and cloning at least one DNA sequence from the library;

(d) genetically altering the cloned DNA sequence;

(e) transforming rose cells with the altered DNA sequence; and

(f) regenerating the genetically altered rose plant from the cells,which plant expresses the altered DNA sequence.

The invention is further directed to a method of isolating plant mRNA,comprising the steps of:

(a) extracting nucleic acids from a sufficient amount of plant tissuestarting material to attain a critical mass amount of RNA forprecipitation;

(b) isolating RNA from the nucleic acids of step (a) using a2-butoxyethanol precipitation technique;

(c) contacting the RNA with a binding partner for mRNA, for exampleoligo-dT or another molecule or entity which has the characteristics ofbinding specifically to mRNA with the exclusion of other forms of RNA orDNA. The binding partner may be immobilized on a solid phase or carrier;this yields immobilized mRNA; and

(d) eluting the immobilized mRNA from the carrier by conventionalelution methods, or obtaining bound mRNA, thereby isolating the mRNAfrom total RNA.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the ethylene biosynthetic pathway including the stepcatalyzed by ACC synthase.

FIG. 2 is a diagram showing the details of steps of cDNA synthesis frommRNA.

FIGS. 3A-3B show the nucleotide sequence of the cDNA clone designatedpRoseKacc7 (SEQ ID NO: 1). The following landmarks are indicated: thestart ATG codon is in bold and underscored; the termination codon of thecoding sequence (TGA) is in bold and double underscored.

FIG. 4 shows the deduced amino acid sequence (SEQ ID NO: 2) encoded bynucleotide sequence SEQ ID NO: 1.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present inventor has isolated, cloned and identified a cDNA sequenceencoding the enzyme ACC synthase in a rose plant (specifically fromRosa). This cDNA sequence corresponds to a gene which is important inthe control of ethylene production. The DNA is expressed in any of anumber of expression systems, including an in vitro expression system toyield a polypeptide product which preferably has ACC synthase enzymaticactivity.

The cloned ACC synthase gene or fragments thereof, when introduced inreverse orientation (antisense) under control of a strong promoter(discussed below in detail), such as the cauliflower mosaic viruspromoter CaMV35S, can be used to genetically modify a rose plant.Selected antisense sequences sharing sufficient homology to ACC synthasegenes in other plants can be used to achieve similar geneticmodification. One result of this modification is a reduction in theamount of translatable ACC synthase-encoding mRNA. As a consequence, theamount of ACC synthase produced in the plant cells is reduced, therebyreducing the rate of conversion of ACC to ethylene. This geneticmodification can effect a permanent change in ethylene levels in themodified plant and be propagated in offspring plants by selfing or otherreproductive schemes. Hence, the invention provides a plant modified asdescribed herein as well as plants which, although modified in adifferent manner achieve similar results or utilize similar concepts asdisclosed herein. The genetically altered plant is used to produce a newvariety or line of plants wherein the alteration is stably transmittedfrom generation to generation.

The rose plant is an ethylene-sensitive flowering plant. A change inethylene level may thus have a great impact on its commercialdesirability. The present invention provides isolated ACC synthase genesobtained specifically from a rose for use in genetic modificationpreferably of rose plants. The full length DNA molecules describedherein are unique to roses and vary significantly in sequence from ACCsynthase DNA in any other unrelated plant species.

Because of such interspecies variation, to achieve stable geneticmodification, it may be important that an ACC synthase gene or genefragment (a) be obtained from the same species or (b) be a functionalderivative of the DNA sequence native to the species. However, it ispossible that a selected sequence from one plant genus or species may beemployed using antisense technology in a different genus or species toachieve a useful effect such as that described here. The presentinvention thus provides for the first time the appropriate DNA sequenceswhich may be used to achieve a stable genetic modification primarily ofrose plants (and of other plants as well).

For the identification, in general, preparation of plasmid DNA,restriction enzyme digestion, agarose gel electrophoresis of DNA,Southern blots, Northern blots after separation of the RNA on aformaldehyde agarose gel, DNA ligation and bacterial transformation werecarried out using conventional methods well-known in the art. See, forexample, Sambrook, J. et al., Molecular Cloning: A Laboratory Manual,2nd Edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1989.

As used herein, the term “plant” refers to either a whole plant, a plantpart, a plant cell, or a group of plant cells. The types of plants whichcan be used in the method of the invention generally includes the genusRosa (roses) which can take up and express the DNA molecules of thepresent invention. It may include plants of a variety of ploidy levels,including haploid, diploid, tetraploid, and polyploid.

A “transgenic plant” is defined as a plant which is genetically modifiedin some way, including but not limited to a plant which has incorporatedheterologous DNA or modified DNA or some portion of heterologous orhomologous DNA into its genome. The altered genetic material may encodea protein, comprise a regulatory or control sequence, or may comprise anantisense sequence or encode an antisense RNA which is antisense to anendogenous DNA or mRNA sequence of the plant. A “transgene” or a“transgenic sequence” is defined as a foreign or atypical gene orpartial sequence which has been incorporated into a transgenic plant.

As used in the present application, the term “substantial sequencehomology” or “substantially homologous” is used to indicate that anucleotide sequence (in the case of DNA or RNA) or an amino acidsequence (in the case of a protein or polypeptide) exhibits substantialfunctional or structural equivalence with another nucleotide or aminoacid sequence. Any functional or structural differences betweensequences having substantial sequence homology will be de minimis; thatis, they will not affect the ability of the sequence to function asindicated in the desired application. Differences may also be simply dueto inherent variations in codon usage among different species. Sequencesthat have substantial sequence homology with the sequences disclosedherein are usually “variants” of the disclosed sequence, such asmutations, but may also be synthetic sequences. Structural differencesare considered de minimis if there is a significant amount of sequenceoverlap or similarity between two or more different sequences or if thedifferent sequences exhibit similar physical characteristics even if thesequences differ in length or structure. Such characteristics include,for example, ability to hybridize under defined conditions, or, in thecase of proteins, immunological crossreactivity, similar enzymaticactivity, etc.

Additionally, two nucleotide sequences are substantially homologous ifthe sequences have at least 70 percent, more preferably 80 percent andmost preferably 90 percent sequence similarity between them. Two aminoacid sequences are substantially homologous if they have at least 50percent, preferably 70 percent, and most preferably 90 percentsimilarity between the active portions of the polypeptides. Further theterm “substantial sequence homology” should be understood to compriseany similarity which meets at least any default criteria of any model(as now readily ascertainable to those of ordinary skill in the art), orany permutation of the following criteria, each of which may also beseparately specified as well to narrow the scope at any time.

A. Search Scoring Models, Parameters, and Costs

(using any program model available, as applicable, including but notlimited to those based upon the Smith-Waterman algorithm (with andwithout extensions or amendments by Gotoh or others), BLAST, PSI-BLAST,Gapped BLAST, or MPSRCH)

Matches: +1, +3, or +5

Mismatches: −1, −3, or −5

Gap Existence Cost (per gap): −1, −6, −10, or −30

Gap Size Cost (per residue): −1, −2, −3, −6, or −10

(such that in some models a gap of length x with a gap existence cost ofA and a gap size cost of B would result in a scoring reduction of A+Bxfor each gap)

B. Lowest Percent Similarity Levels

(defined as the above scoring divided by any of: the length of thesequence at issue, the length of the local portion of the sequence atissue, or the average length of the sequence at issue and the comparedsequence)

for Amino Acid Sequence Comparisons

all percentages from 50% to 100% in 2% increments (based on eitherglobal similarities or local similarities)

for Nucleotide Sequence Comparisons

all percentages from 70% to 100% in 2% increments (based on eitherglobal similarities or local similarities)

The term “hybridization” as used herein is generally understood to meanhybridization at appropriate conditions of stringency as would bereadily evident to those skilled in the art depending upon the nature ofthe probe sequence and target sequences. Conditions of hybridization andwashing are well-known in the art, and the adjustment of conditionsdepending upon the desired stringency by varying incubation time andtemperature and ionic strength of the solution are readily accomplished.See, for example, Sambrook, J., et al., Molecular Cloning: A LaboratoryManual, Second Edition, Cold Spring Harbor Press, Cold Spring Harbor,N.Y. (1989). The choice of conditions is dictated by the length of thesequences being hybridized, in particular the length of the probesequence, the relative G-C content of the nucleic acid and the amount ofmismatches to be permitted. Low stringency conditions are preferred whenpartial hybridization between strands that have lesser degrees ofcomplementarity is desired. When perfect or near-perfect complementarityis desired, high stringency conditions are preferred. For typical highstringency conditions, the hybridization solution contains 6× SSC, 0.01MEDTA, 5× Denhardt's solution and 0.5% SDS. Hybridization is carried outat about 68° C. for 3-4 hours for fragments of cloned DNA and 12-16hours for total eukaryotic DNA. For lower stringency, the temperature isreduced to about 12° C. below the melting temperature (T_(m)) of theduplex. The T_(m) is known to be a function of G-C content and duplexlength as well as the ionic strength of the solution.

By “functional derivative” of a nucleic acid (or poly- oroligonucleotide) is meant a “fragment,” “variant,” “homologue” or“analogue” of the gene or DNA sequence encoding ACC synthase, or in someway related to the production or use of ACC synthase, especially roseACC synthase. A functional derivative may retain at least a portion ofthe function of the ACC synthase-encoding DNA which permits its utilityin accordance with one embodiment of the present invention. Suchfunction may include the ability to hybridize with native rose orhomologous DNA from another plant which encodes ACC synthase or with anmRNA transcript thereof, or, in antisense orientation, to inhibit thetranscription and/or translation of rose ACC synthase mRNA or the like.

A “fragment” of the gene or DNA sequence refers to any subset of themolecule, that is, a shorter polynucleotide- or oligonucleotide. A“variant” refers to a molecule substantially similar to either theentire gene or a fragment thereof, such as a nucleotide substitutionvariant having one or more substituted nucleotides but which maintainsthe ability to hybridize with the particular gene or to encode a mRNAtranscript which hybridizes with the native DNA. A “homologue” refers toa fragment or variant sequence from a different plant genus or species.An “analogue” refers to a non-natural molecule substantially similar toor functioning in relation to either the entire molecule, the variant,or to a fragment thereof.

“Altered” expression” or an “alteration” of expression of a gene (mostparticularly of ACC synthase), as used herein, refers to any process orresult whereby the normal expression of the gene, for example thatoccurring in an “unmodified” rose plant, defined as a known,conventional, naturally-occurring rose plant, is changed in somefashion. As intended herein, an alteration is a complete or preferably apartial reduction in the expression of ACC synthase, but may alsoinclude a change in the timing of expression, or another state whereinthe expression of ACC synthase differs from that which would be mostlikely to occur naturally in an unmodified rose plant, variety orcultivar. A preferred alteration is one which results in a decrease inethylene production by the plant compared to ethylene production in anunmodified plant.

In producing a genetically altered plant according to this invention, itis preferred to select individual plantlets or plants by the desiredtrait, generally reduced ACC synthesis expression and reduced ethyleneproduction. Expression of ACC synthase can be measured by quantitatingthe amount of ACC synthase mRNA using conventional hybridizationtechniques. Alternatively, the amount of ACC synthase protein can bequantitated, for example in a conventional immunoassay method using aspecific antibody such as those described herein. Finally, the ACCsynthase enzymatic activity can be measured using biochemical methods asdescribed in Kionka et al., supra; Amrhein et al., supra; or Hoffman N.E., et al., supra. Ethylene biosynthesis in the plantlet or plant can bequantitated using known methods Yang, S. F. et al. (1984), Annu. RevPlant Physiol:35, 155-189); Abeles, F. B. et al eds, Ethylene in PlantBiology, Academic Press, New York, 1976 White, J. W., ed., Geranium IV.The Growers Manual, Edition Four, Ball Publishing, Geneva, Ill.

In order for a newly inserted gene or DNA sequence to be expressed,resulting in production of the protein which it encodes (or, in the caseof antisense DNA, to be transcribed, resulting in an antisense RNAmolecule), the proper regulatory signals should be present in the properlocation with respect to the coding or antisense sequence. Theseregulatory signals may include a promoter region, a 5′ non-translatedleader sequence and a 3′ polyadenylation sequence as well as enhancersand other known regulatory sequence. The promoter is a DNA sequence thatdirects the cellular machinery to transcribe the DNA to produce RNA. Thepromoter region influences the rate at which the mRNA product and, ifthe DNA encodes a protein, the resultant protein product, are made. The3′-polyadenylation signal is a non-translated region that functions inplant cells to cause the addition of a polyadenylate stretch to the 3′end of the mRNA to stabilize it in the cytoplasm for subsequenttranslation.

A promoter DNA sequence is operably linked to a second DNA sequence andregulates its transcription. If the second DNA sequence encodes aprotein, the promoter DNA sequence is said to be “operably linked” if itaffects the transcription of the mRNA encoding the protein product fromthe second DNA sequence. A DNA sequence comprising a promoter isgenerally physically near the coding sequence in the same recombinantconstruct, though physical contiguity is not required. “Strong”promoters are able to direct RNA synthesis at higher rates than weakerpromoters. Certain promoters direct RNA production at higher levels onlyin particular types of cells and tissues. Promoters that direct RNAproduction in many or all tissues of a plant without the need for“induction” by a specific inducer substance are called constitutivepromoters. The operation of a constitutive promoter is relativelyindependent of the developmental stage of the cell in which it iscontained and is most preferred for the present invention. An induciblepromoter is one which, in response to the presence of an inducer, isactivated. Hence, a coding sequence driven by an inducible promoter canbe turned on or off by providing or withdrawing the inducer. A promotermay be homologous, derived from the same species as the coding sequence.Preferably, the promoter is heterologous, that is, derived from anotherspecies, or even from a virus.

Expression levels from a promoter which is useful for the presentinvention can be tested using conventional expression systems, forexample, by measuring levels of a reporter gene product (protein ormRNA) in extracts of the leaves, stems, roots and flowers of atransgenic plant into which the promoter/reporter have been introduced.

Cauliflower mosaic virus (CaMV) is a double-stranded DNA plant virus. Itcontains two promoters responsible for the production of transcripts of35S and 19S in size in infected plants (Guilley, H., et al, Cell 30:763(1982)). The 35S promoter (CaMV35S) is one of the strongest constitutiveheterologous promoters known in plants (Odell, et al., Nature313:810-812 (1985); Jensen, et al., Nature 321:669-674 (1986);Jefferson, et al., EMBO J. 6:3901-3907 (1987); Kay, et al., Science236:1299-1302 (1987); Sanders, et al., Nucl. Acids Res. 4:1543-1558(1987)). Two different domains within the CaMV 35S promoter maydifferentially regulate expression of a coding sequence in differentplant tissues (domain A, from nucleotides −90 to +8) vs. domain B fromnucleotides −343 to −90), as described by Benfey, et al., 1989 EMBO J8:2195-2202.) The CaMV35S promoter is active in isolated protoplasts(Fromm, M., et al., Proc. Natl. Acad. Sci. USA 82:5824 (1985)) and isexpressed in all organs of various transgenic plants in the absence ofany viral protein, making it widely used in plant genetic engineering.

Because of variability in the expression of genes driven by the CaMV35Spromoter, (which may be either an intrinsic property of the promoter ora result of variability in the position at which CaMV35S promoter-drivenDNA sequence is integrated into the genome of the transformed plant),CaMV35S may be particularly useful for effecting different degrees ofaltered gene expression by an antisense sequence which the promotercontrols. Additional useful plant promoters in, for example, othercaulimoviruses (a group of double-stranded DNA viruses to which thecauliflower mosaic virus belongs) have also been developed and areuseful for similar applications. Two caulimoviruses distantly related toCaMV are the figwort mosaic virus (FMV) (Richins, et al., Nucl. AcidsRes. 15:8451-8466 (1987)) and the carnation etched ring virus (CERV)(Hull, et al., EMBO J 5:3083-3090 (1986). The promoters of FMV and CERVwhich are homologues of the CaMV35S promoter are described in Rogers,U.S. Pat. No. 5,378,619. Any of the foregoing viral promoters, as wellas other viral promoters which act as strong promoters for expression ofplant DNA sequences in plant cells, may be used to drive the expressionof the DNA molecules of the present invention.

Certain other strong plant promoters are also useful to direct theexpression of the ACC synthase DNA (or antisense sequences) of thepresent invention. For example, the small subunit (SSU) of the enzymeribulose-1,5-bisphosphate carboxylase (RuBPCase), the primary enzyme ofthe carbon fixation pathway in chloroplasts of plants of the C3 class isan example of a polypeptides known to be highly expressed in plants. Ahighly efficient SSU promoter DNA such as the promoter DNA from the SSUgene denominated SSU301 from Petunia (Bedbrook, et al., U.S. Pat. No.4,962,028) may be used herein. The promoter may be used in the form ofan isolated 5′ fragment of the SSU gene, and preferably has the 3′ endof the fragment modified to create a restriction site which permitsready fusions with the ACC synthase antisense DNA of the presentinvention. The promoter may be conveniently arranged to form anexpression cassette comprising a 5′ fragment (the promoter region of theSSU gene), a 3′ fragment and a linker region connecting the twofragments. The fusion points between the 5′ fragment and the linkerregion and between the 3′ fragment and the linker region are preferablymodified to create restriction sites which permit the antisense DNA ofthe present invention to be substituted for the linker so as to yield“chimeric” genes containing the complete proximal 5′ and 3′ regions ofthe SSU gene but none of the SSU coding sequence

Other plant promoter enhancer/sequences which may be used in accordancewith the present invention have been described in the followingreferences: Coruzzi, et al., 1984, EMBO J. 3:1671-1680;Herrera-Estrella, et al., 1984, Nature 310:115-120; Apel, et al., 1978,Eur. J. Became. 85:581-588; Stiekema, et al., 1983, Plant Physiol.72:717-724; Thompson, et al., 1983, Planta 158:487-500; Jones, et al.,1985, EMBO J. 4:2411-2418; Stockhaus, et al., 1989, Plant Cell1:805-814; Gurley, et al., 1986, Mol. Cell Biol. 6:559-565; Landsmann,et al., 1988, Mol. Gen. Genet. 214:68-73; Bevan, et al., 1989, EMBO J.8:1899-1906; Benfey, et al., 1989, Science 244:174-181.

Additionally, certain bacterial promoters have been observed to beexpressed in plants, including the Rhizobium meliloti FIXD gene promoter(Puhler, et al., U.S. Pat. No. 4,782,022) and the nopaline synthasepromoter (Ha, et al., 1989, Nucl. Acids Res. 17:215-224; An et al.,1988, Plant Physiol. 88:547-552). Several promoter sequences, termed therol A, B and C promoters, have been identified in Agrobacteriumrhizogenes (Schmulling, et al., 1989, Plant Cell 1:665-670; Sugaya, etal., 1989, Plant Cell Physiol. 30:649-654).

To test the activity of a promoter, E. coli β-glucuronidase (GUS) codingsequence or a mutant Arabidopsis EPSP synthase gene which encodes anenzyme tolerant of glyphosate herbicides may be used as a reporter gene.Transformed plant cells or plants containing the GUS gene operablylinked to the promoter being tested are assayed using a histologicalstaining procedure to determine GUS activity in the transformed cells.

The present invention provides antisense oligonucleotides andpolynucleotides complementary to the gene or genes encoding ACC synthasein a rose plant. Such antisense oligonucleotides, should be at leastabout six, ten, twenty, or fifty nucleotides in length to provideminimal specificity of hybridization, and may be complementary to onestrand of DNA or to mRNA encoding ACC synthase (or to a portionthereof), or to flanking sequences in genomic DNA which are involved inregulating ACC synthase gene expression. The antisense oligonucleotidemay be as large as about 100 nucleotides, an may extend in length up toand beyond the full coding sequence for which it is antisense. Theoligonucleotides can be DNA or RNA or chimeric mixtures or derivativesor modified versions thereof, single-stranded or double-stranded.

The action of the antisense nucleotide may result in specificalteration, primarily inhibition, of ACC synthase gene expression incells. For a general discussion of antisense, see: Alberts, B., et al.,MOLECULAR BIOLOGY OF THE CELL, 2nd Ed., Garland Publishing, Inc., NewYork, N.Y. (1989), in particular, pages 195-196, which reference ishereby incorporated by reference.

The antisense oligonucleotide may be complementary to any portion of theACC synthase encoding sequence. In one embodiment, the antisenseoligonucleotide may be between about 6, 10, 20, or 50 and 100nucleotides, and may be complementary to the initiation ATG codon and anupstream, non-coding translation initiation site of the ACC synthasesequence. For example, antisense nucleotides complementary primarily fornon-coding sequence, are known to be effective inhibitors of theexpression of genes encoding transcription factors (Branch, M. A., 1993Molec. Cell. Biol. 13:4284-4290).

Preferred antisense oligonucleotides are complementary to a portion ofthe mRNA encoding ACC synthase. For instance, it is expected that byintroducing a full length cDNA clone gene in an antisense orientation,successful alteration of gene expression will be most probable.Naturally, introduction of partial sequences, targeting to specificregions of the gene, and the like can be effective as well. An exampleof a preferred antisense oligonucleotide for a rose is a 50mer which isantisense to 50 nucleotides in the 5′ half of an RNA transcript of anACC-encoding cDNA (such as SEQ ID NO: 1), more preferably any stretch of50 nucleotides in the first 500 nucleotides of the 5′ part of the RNAtranscript. For example, the antisense oligonucleotide can be antisenseto nucleotides 1-50, 2-51, 3-52, 4-53, 5-54, etc., of the RNAtranscript. Alternatively, the antisense oligonucleotide can be shorterfor wither plant, for example a 30-mer, and be antisense to any 30nucleotide stretch of the RNA transcript, preferably in the first 500 5′nucleotides.

As is readily discernible by one of ordinary skill in the art, theminimal amount of homology required by the present invention is thatsufficient to result in sufficient complementarity to providerecognition of the specific target RNA and inhibition or reduction ofits translation or function while not affecting function of other mRNAmolecules and the expression of other genes. While the antisenseoligonucleotides of the invention comprise sequences complementary to atleast a portion of an RNA transcript of ACC synthase, absolutecomplementarity, although preferred, may not be required. A sequence“complementary to at least a portion of” another sequence, as referredto herein, may have sufficient complementarity to be able to hybridizewith that of other sequences in vivo, perhaps forming a stable duplex.Naturally, the ability to hybridize may depend on both the degree ofcomplementarity and the length of the antisense nucleic acid. Generally,the longer the hybridizing nucleic acid, the more base mismatches withthe ACC synthase target sequence it may contain and still form a stableduplex. One skilled in the art can ascertain a tolerable degree ofmismatch by use of standard procedures to determine the meltingtemperature of the hybridized complex as discussed above and othertechniques.

The antisense RNA oligonucleotides may be generated intracellularly bytranscription from exogenously introduced nucleic acid sequences. Thus,antisense RNA may be delivered to a cell by transformation ortransfection or infection with a vector, such as a plasmid or a virus,into which is incorporated (a) DNA encoding the antisense RNA andoperably linked thereto (b) the appropriate regulatory sequences,including a promoter, to express the antisense RNA in a target host cell(and whole plant). Within the cell the exogenous DNA or a portionthereof may be transcribed, producing an antisense RNA of the invention.Vectors can be plasmid, viral, or others known in the art which are usedfor replication and expression in plant cells. Expression of thesequence encoding the antisense RNA can be by any promoter known in theart to act in a plant, preferably rose cells. Such promoters can beinducible or preferably are constitutive as described above. Such avector, preferably a plasmid, becomes chromosomally integrated such thatit can be transcribed to produce the desired antisense RNA. Such plasmidor viral vectors can be constructed by recombinant DNA technologymethods that are standard in the art.

An oligonucleotide, between about 6 and about 100 bases in length andcomplementary to the target sequence of ACC synthase, as described abovemay be prepared by chemical synthesis from mononucleotides or shorteroligonucleotides, or produced by recombinant means.

Basic procedures for constructing recombinant DNA and RNA molecules inaccordance with the present invention are disclosed by Sambrook, J., etal., In: Molecular Cloning: A Laboratory Manual, Second Edition, ColdSpring Harbor Press, Cold Spring Harbor, N.Y. (1989), which reference isherein incorporated by reference. Oligonucleotide molecules having astrand which encodes antisense RNA complementary to an ACC synthasesequence can be prepared using procedures which are well known to thoseof ordinary skill in the art. Details regarding such procedures aredescribed in: Belagaje, R., et al., J. Biol. Chem. 254:5765-5780 (1979);Maniatis, T., et al., In: MOLECULAR MECHANISMS IN THE CONTROL OF GENEEXPRESSION, Nierlich, D. P., et al., eds., Acad. Press, N.Y. (1976); Wu,R., et al., Prog. Nucl. Acid Res. Molec. Biol. 21:101-141 (1978);Khorana, H. G., Science 203:614-625 (1979)). Automated synthesizers maybe used for DNA synthesis (such as are commercially available fromBiosearch, Applied Biosystems, etc.).

Techniques of nucleic acid hybridization are disclosed by Sambrook etal. (supra), and by Haymes, B. D., et al., In: NUCLEIC ACIDHYBRIDIZATION, A PRACTICAL APPROACH, IRL Press, Washington, D.C.(1985)), which references are herein incorporated by reference.

The transgenic plants of the present invention may be prepared by DNAtransformation using any method of transformation known in the art.These methods include transformation by direct infection orco-cultivation of plants, plant tissue or cells with Agrobacteriumtumefaciens (Horsch, et al., Science 225:1229 (1985); Marton, CellCulture and Somatic Cell Genetic of Plants 1:514-521 (1984)); Fry, etal., Plant Cell Reports 6:321-325 (1987); direct gene transfer intoprotoplasts or protoplast uptake (Paszkowski, et al., EMBO J. 12:2717(1984); Loerz, et al., Mol. & Gen. Genet. 178:1199 (1985);electroporation Fromm, et al., Nature 319:719 (1986)); microprojectileor particle bombardment (Klein, et al., Bio/Technology 6:559-563(1988)); injection into protoplasts cultured cells and tissues (Reich etal., Bio/Technology, 4:1001-1004 (1986)); or injection into meristematictissues of seedlings and plants (De La Pena, et al, Nature, 325:274-276(1987); Graves, et al., Plant Mol. Biol. 7:43-50 (1986); Hooykaas-VanSlogteren, et al., Nature 311:763-764 (1984); Grimsley, et al.,Bio/Technology 6:185 (1988); and Grimsley, et al., Nature 325:177(1988).

The Agrobacterium tumefaciens strain 208 carrying the disarmed pMP90RKplasmid can be used to achieve transformation. Used for planttransformations, the vector plasmid may be introduced into theAgrobacterium by the triparental conjugation system (Ditta, et al.,(1980) Proc. Natl. Acad. Sci. USA 77:7347-7451) using the helper plasmidpRK2013. The vectors may be transferred to plant cells by the virfunctions encoded by the disarmed pMP90RK Ti plasmid. The vector isopened at the pTiT37 right border sequence and the entire vectorsequence is inserted into the host plant chromosome. The pMP9ORK Tiplasmid is probably not transferred to the plant cell but remains in theAgrobacterium.

Normally, regeneration will be involved in obtaining a whole plant fromthe transformation process. The term “regeneration” as used herein,means growing a whole plant from a plant cell, a group of plant cells, aplant part or a plant piece (e.g. from a protoplast, callus, tissuepart, or explant, etc.) Plant regeneration from cultured protoplasts isdescribed in Evans, et al., Handbook of Plant Cell Cultures 1:124-176(MacMillan Publishing Co. New York 1983); Davey, M. R., Protoplasts(1983), Lecture Proceedings, pp.12-29, Birkhauser, Basel, 1983); P. J.Dale, ibid, at pp. 31-41, (Birkhauser, Basel 1983); and H. Binding,Plant Protoplasts, pp.21-73, CRC Press, Boca Raton, 1985).

Plant parts obtained from the regenerated plant in which expression ofan ACC synthase gene has been altered, such as flowers, seeds, leaves,branches, fruit, and the like are included within the definition of“plant” as stated above, and are included within the scope of theinvention. Progeny and variants and mutants of the regenerated plantsare also included, especially if these parts comprise the introduced DNAsequences.

The present invention also provides ACC synthase proteins encoded for bythe cDNA molecules described above. For roses, such proteins preferablyhave the amino acid sequence of SEQ ID NO: 2 as shown in FIG. 4. In eachcase, these proteins, or functional derivatives thereof, are preferablyproduced by recombinant methods optionally in combination with chemicalmethods.

A “functional derivative” of the ACC synthase protein is a “fragment,”“variant,” “analog,” or “chemical derivative” of ACC synthase, whichretains at least a portion of the function of the ACC synthase whichpermits its utility in accordance with the present invention. Suchfunction includes enzymatic activity or immunological crossreactivitywith an antibody specific for ACC synthase. A fragment of the ACCsynthase protein refers to any subset of the molecule, that is, ashorter peptide. A variant refers to a molecule substantially similar toeither the entire protein or a fragment thereof. Variant peptides may beconveniently prepared by direct chemical synthesis using methodswell-known in the art. An “analog” of ACC synthase refers to anon-natural protein substantially similar to either the entire proteinor a fragment thereof. A chemical derivative of ACC synthase containsadditional chemical moieties not normally a part of the protein orpeptide fragment thereof. Covalent modifications of an ACC synthasepeptide are included within the scope of this invention. Suchmodifications may be introduced into the molecule by reacting targetedamino acid residues of the peptide with an organic derivatizing agentthat is capable of reacting with selected side chains or terminalresidues.

A protein or peptide according to the present invention may be producedby culturing a cell transformed with a DNA sequence of this invention,allowing the cell to synthesize the protein, and obtaining the proteinfrom the culture medium if it is secreted, or if it is intracellular,obtaining it by extraction. In a preferred embodiment, the protein isproduced in a cell free system, for example, as described by Ranu, R.S., et al, 1979, Meth. Enzymol. 60:459-484 and Ranu, R. S., et al,(1996) Gene Expression 5:143-153.

To produce an isolated, purified protein or peptide, the in vitrotranslation product or the cell or tissue extract from transformed plantcells or plant parts is subjected to conventional biochemicalpurification methods, including but not limited to affinitychromatography using an antibody specific for an epitope of the protein.

Having now generally described the invention, the same will be morereadily understood through reference to the following examples which areprovided by way of illustration, and are not intended to be limiting ofthe present invention, unless specified.

EXAMPLES

Plant Material

Rosa (rose) plants grown and maintained in a greenhouse were used toclone the cDNA corresponding to ACC synthase genes. Flower tissue in theform of senescing flower petals (from different stages) were collectedin liquid nitrogen and used immediately or stored at −70° C. until use.

Messenger RNA (mRNA) Isolation

The quality of the mRNA largely determines the quality of cDNA librarygenerated subsequently for cDNA cloning of ACC synthase genes. By“quality of the mRNA” is intended the presence of all the desired mRNAspecies, especially those mRNA molecules that are present in cells inrelatively low abundance (either because of the number of gene copies,the rate of transcription or the stability of the mRNA). The most widelyused method for preparation of RNA utilizes extraction with 4 Mguanidine thiocyanate of total RNA (Chomczynski, P., et al. (1987),Anal. Biochem.. 162:156-159). When this method was tried by the presentinventor for a geranium, the quality of RNA obtained was inadequate anddid not permit a generation of a useable, high quality cDNA library(containing cDNA inserts corresponding to the least abundant mRNAs).Thus, when cDNA libraries prepared using the conventional method werescreened for the presence of cDNA inserts encoding ACC synthase, theclones identified contained only partial genes or, mostly frequently,false positives. This problem alone made the process of isolating theACC synthase genes of this invention extremely difficult andchallenging. This conclusion was also suggested from the results ofexpression screening of such libraries with antibodies specific for theACC synthase protein. In sum, the prior art RNA isolation technique atbest invited experiments to try to find the full length genes, butprovided no reasonable expectation of success. Problems posed by thepoor quality of the total RNA prepared using conventional methods ledthe present inventor to look for alternative means for obtaining RNA ofsufficiently high quality to be useful for the purposes of thisinvention, namely preparation of a cDNA library having a highprobability of including a full length DNA sequences corresponding tolow-abundance mRNAs, in particular full-length ACC synthase codingsequences.

Preparation of RNA

The preferred method discovered by the present inventor was based on theprecipitation of RNA from a tissue extract using 2-butoxyethanol(Manning, K., 1991, Anal. Biochem. 195:45-50) with modifications. Thismethod is referred to herein as “a 2-butoxyethanol precipitationtechnique.” This technique was originally developed for RNA isolation,and by adapting it for mRNA isolation, the extraordinary results of thisinvention were achieved. Generally, in order to achieve the required RNAprecipitation, a co-precipitation critical mass of RNA must be presentin the preparation. The relative low proportion of RNA in relation tothe total extracted material required the recognition by the presentinventor that the standard amount of tissue extract used in RNApreparation, about 1 gram or less, would be insufficient for certaintypes of plants such as roses (discussed more fully below). The successdescribed herein was ultimately attained by using an unusually largeamount of tissue. For effort with roses, this was about 3-5 grams.While, in hindsight, this may seem like a simple problem and solution,in fact, this problem does not appear to have been considered by others,and, therefore, the novel method is not an obvious modification of theolder technique.

This problem in part stems from the fact that the desired precipitationis “non-linear,” meaning that no simple linear relationship existsbetween the mass of RNA and the amount of precipitation. Rather, theprocess is a threshold phenomenon, and unless that critical mass ispresent, precipitation will not occur. For these reasons, the prior arttechnique would appear on its face to be inapplicable for obtaining ahigh quality mRNA preparation from woody plants such as geraniums orroses. Surpassing such a critical amount of RNA, that is, an amount atwhich precipitation occurs, permitted the method, as modified, todemonstrate its full utility. Hence, the present inventor achieved anunexpected and extraordinary result, in spite of the fact that thetechnology underlying the modifications introduced to earlier methodshad been available. Those of ordinary skill in the art may haveappreciated (although this is not evident) that a key impediment was inthe obtaining of high quality mRNA to generate a fully representativecDNA library. Furthermore, a long felt need in the art for such alibrary had not been satisfied. Nevertheless, substantial attempts inthe prior art failed because practitioners did not understand the truenature of the reasons for failure of this type of technique.

The present inventor's discovery of a means to here achieve theco-precipitation critical mass of RNA is particularly important to theclass of plants which have a low proportion of RNA in their tissue, suchas less than only {fraction (1/10,000)}th of the total tissue usuallyobtained. It is also particularly important for woody plants such asgeraniums or roses, for which the present invention is particularlyuseful. These groups of plants comprises plant species that have a lowproportion of RNA in their tissue relative to non-nucleic acid material.This is in contrast to other plants which have a higher proportion ofRNA and are amenable to the preparation of high quality mRNA (and cDNAcorresponding thereto) by the traditional approaches of the prior art.While this “low RNA” group of plants is known to include at leastPelargonium and Rosa (rose) species, it is clear that other plants alsofall in this category, as would be evident to those skilled in the art.This group of plants is characterized in one manner as being woody (thatis, they contain large amounts of fiberous material) and thereforehaving a low relative abundance of RNA, or conversely, as a highrelative proportion of non-nucleic acid material. Thus, in this categoryof low RNA plants, it would be necessary to use a “large” amount oftissue, namely, an amount which (depending upon the particular plant ortechnique) is sufficient to yield a co-precipitant critical mass oftotal RNA in the process. For Pelargonium and Rosa, and the like, aco-precipitant critical mass of RNA is about 200 μg for successfulimplementation of the 2-butoxyethanol precipitation technique describedherein. (Other RNA isolation techniques or plants may, of course, eachhave their own critical mass, that is, the presence of enough total RNAfor precipitation to actually occur.) Thus, for the present techniqueand plants, about 3-5 grams of flower tissue was used initially. Thismay represent a minimum amount for some plants. Naturally more wouldalso work.

The flower tissue was ground into a powder using a pestle and mortarprecooled by liquid nitrogen. The resulting material was then groundwith 12-20 ml of extraction buffer (0.2M boric acid/Tris-HCl and 10 mMEDTA (pH 7.6)), followed by addition of 0.24-0.4 ml of 25% sodiumdodecyl sulfate (SDS) and 0.24-0.4 ml of 2-mercaptoethanol (2-ME).

The mixture was brought to room temperature and extracted with an equalvolume of extraction buffer, saturated phenol/chloroform mixture. Themixture was centrifuged at 20,000×g at room temperature. The upperaqueous phase was collected and kept in a fresh tube. The interphase andlower organic phase were re-extracted with an equal volume of extractionbuffer containing SDS and 2-ME. After centrifugation at 20,000×g, thesecond aqueous phase was removed and combined with the first aqueousphase. The pooled aqueous phase was diluted with 2.5 volume of water anda quantity of 1M sodium acetate (pH 4.5) sufficient to make the finalconcentration 80 mM.

This was followed by addition of 0.4 volumes of 2-butoxyethanol (2-BE).After 30 minutes on ice, the mixture was centrifuged at 20,000×g for 10minutes at 0° C. The clear supernatant was collected. Additional 2-BEwas added to bring the total to one volume. After 30 minutes on ice, thenucleic acid-containing pellet was collected by centrifugation at20,000×g for 10 minutes at 0° C. The pellet was washed first with a 1:1(v/v) mixture of extraction buffer and 2-BE, followed by 70% ethanolcontaining 0.1M potassium acetate (pH 6.0), and finally with 100%ethanol. The pellet was then air dried.

The nucleic acid pellet was dissolved in water to a concentration ofabout 1 mg/ml and sufficient 12M LiCl was added to bring the LiClconcentration to 3M. After one hour on ice, an RNA precipitate wascollected by centrifugation at 12,000×g for 10 minutes at 0° C. Thepellet was washed twice with 3M LiCl and once with 70% ethanol and wasfinally air dried. RNA was dissolved 0.2-0.5 ml of 10 mM Tris-HCl, 1 mMEDTA (pH 8.0) (TE buffer).

Isolation of mRNA

PolyA⁺mRNA was isolated by binding to Dynabeads-oligo(dT)25 (Dynal,Inc., Lake Success, N.Y.). The oligo (dT)25 is a preferred bindingpartner, in addition others are known in the art, the key function beingmerely the ability to selectively attach to the mRNA. For this bindingpartner, the protocol provided by the manufacturer was used. PolyA⁺RNAwas bound to Dynabeads in the presence of 1× binding buffer for 30minutes. The Dynabeads serve as one of the many possible solid phasesupports or carriers. This served to immobilize the mRNA. The beads werewashed three times with washing buffer containing lithium dodecylsulfate (LiDS) and once with wash buffer alone. mRNA was eluted from thebeads with 50 μl of TE buffer.

The composition of the buffers was as follows:

(a) 1× Binding Buffer: 10 mM Tris-HCl (pH 7.5), 0.5M LiCl, 1 mM EDTA,0.5% LiDS;

(b) Washing Buffer with LiDS: 10 mM Tris-HCl, 0.15M LiCl, 1 mM EDTA,0.1% LiDS

Synthesis of cDNA

The mRNA preparation (5 μg) isolated as above was used to synthesizecDNA using the ZAP Express® cDNA synthesis system from Stratagene (LaJolla, Calif.). The details of the steps of synthesis are presented inFIG. 2. The first strand synthesis was carried out with murine-Moloneyleukemia virus reverse transcriptase (M-MuLV-RT) in the presence ofmRNA, a primer containing a 50 base long oligonucleotide

5′-GAGAGAGAGAGAGAGAGAGAACTAGTCTCGAGTTTTTTTTTTTTTTTTTT-3′ [SEQ ID NO: 3]                             XhoI

with an XhoI restriction recognition site (shown underscored). Thisallows the finished cDNA to be inserted into the ZAP Express® Vector inthe sense orientation (EcoRI-XhoI) with respect to the LacZ promoter.The poly(dT) region binds to the poly(A) tail of mRNA template and thereverse transcriptase starts the synthesis of first strand. Thenucleotide mixture for the synthesis of first strand contained dATP,dGTP, dTTP, and 5-methyldCTP. The first strand has methyl groups on eachcytosine base which protects cDNA from restriction enzymes used insubsequent cloning steps.

RNase H nicks the RNA bound to the first strand cDNA to produce multiplefragments which serve as primers for DNA polymerase I (PolI). PolInick-translates the RNA fragments into second strand of cDNA. The cDNAends are blunted in the presence of Klenow fragment and dNTPs. The EcoRIadaptors as shown below

5′AATTCGGCAGAG-3′ [SEQ ID NO: 4]    GCCGTCTCp5′

are ligated to the blunt ends. The XhoI digestion of cDNA releases theEcoRI adaptor and residual linker-primer from 3′-end of the cDNA. ThecDNA is size fractionated on Sephacryl-S400® and then ligated to the ZAPExpress Vector® arms.

Only cDNA of 1.5 kb pairs was used to ligate into ZAP Express Vector®and then packaged into bacteriophages using Gigapack® III Gold Packagingextract protocol from Stratagene. The unamplified cDNA library generatedin this way was used for subsequent screening for ACC synthase genes.

Development of a Polymerase Chain Reaction (PCR) Probe for the Screeningof ACC Synthase Genes.

The first strand of cDNA synthesis was carried out with 2 μg of mRNAusing the ready-to-go T-Primed First-Strand synthesis protocol obtainedfrom Pharmacia Biotechnology (Piscataway, N.J.). The first strand cDNAproduct was then used to develop a PCR probe. PCR amplification (Mullis,K. B., et al, F. A. (1987), Meth. Enzymol. 155:355-350) was performed ina Techne PHC-2 Thermocycler (Techne, Princeton, N.J.).

The following PCR primers were used for the Rose effort:

Primer I

5′-GGIC/TTICCIGGITTC/TC/AGIG/ATIGG-3′

This is alternately designated as

5′GGNYTNCCNGGNTTYMGNRTNGG3′ (where N=inosine) [SEQ ID NO: 5]

Primer II

5′-CAIAIICG/TG/AAAG/CC/AAICCIG/AGICC/TTC-3′

This is alternately designated as:

5′ CANANNCKRAASMANCCNRSYTC3′ (where N=inosine) [SEQ ID NO: 6]

The PCR reaction (50 μl) contained 5 mM Tris-HCl (pH 8.3); 3 mM MgCl₂,50 mM KCl, 50 pmol of primer I: 3 μl of synthesized first strand cDNA,200 mM each of the four dNTPs and 25 units of- (DELTA) Taq DNApolymerase (Amersham Life Sciences, Inc., Arlington Heights, Ill.).Reaction samples were overlaid with 20 μl of mineral oil. After aninitial denaturation at 95° C. for 4 minutes, samples were subjected totwo cycles in which conditions were 94° C. for one minute fordenaturation, 60° C. for two minutes for annealing; and 72° C. for oneminute for extension. It was followed by 30 cycles at 94° C. for 30seconds; 60° C. for one minute; and 72° C. for 45 seconds. The lastcycle was at 72° C. for 5 minutes.

On analysis by agarose gel electrophoresis, the amplified DNA showed aDNA band of about 360 bp. The band was localized in the gel under a UVlamp and excised. DNA from the gel was purified by using Spin-BindRecovery system from FMC BioProducts (Rockland, Me.). The DNA was thencloned using the protocol provided by manufacturer into a TA CloningVector called pCRII (Invitrogen, San Diego, Calif.) and then sequenced.

The 360 bp fragment cloned in the pCRII vector was excised and used toprepare a [³²P]-labeled probe. The Maga Prime system from Amersham LifeScience, Inc. (Arlington Heights, Ill.) was used according to themanufacturer's protocol. The labeled DNA probe incorporated nearly 70%of the input [α³² P]dATP.

Isolation of cDNA Clones from the cDNA Library

Unamplified recombinant bacteriophages (1×10⁶ pfu) were screened withthe [³²P] labeled probe. Phages (50,000 pfu) were grown on a 150-mm NZYplate for six hours at 37° C. The plates were cooled to 4° C. Phageswere transferred onto a Hybond-N+ nylon membrane (Amersham, Inc.) for 40seconds. The DNA on membrane was denatured by treatment with 1.5 MNaCl-0.5M NaOH for 2 minutes, neutralized in 1 .5M NaCl-0.5M Tris-HCl(pH 8.0) for 5 minutes and finally washed in 0.2 M Tris-HCl (pH 7.5),2×SSC for 30 seconds. DNA was fixed onto the membrane by UVcross-linking (Strategene UV Cross-Linker) and then baked at 80° C. forone hour.

The membrane was treated with Rapid-hyb® buffer (Amersham, Inc.) at 55°C. for one hour for prehybridization and then probed with [³²P]-labeledPCR probe for 3 hours at 55° C. The membranes were washed with2×SSC-0.1% SDS for one hour at room temperature and with 0.2-x SSC-0.1%SDS at room temperature. The filters were then exposed to X ray film(Fuji).

A total of 33 putative clones were identified during the first screeningof the cDNA library. Of these putative clones, eight were furtherscreened in the second screening cycle at lower density (1000-4000 pfu).Eight putative clones from the second screening were subjected to atertiary screening. All these eight clones showed strong signal and werejudged to be positive.

These clones were in vivo excised out of the pBK-CMV phagemid vector,and the size of the cDNA insert (representing ACC synthase genes) wasdetermined by electrophoresis. Clones were judged to be nearlyfull-length, as confirmed by subsequent DNA sequencing.

DNA Sequencing of Clones

The dideoxy chain termination method (Sanger, F., et al., (1977), Proc.Natl. Acad. Sci. USA 74:5463-5467)) was used to sequence the ACCsynthase cDNA clones for a rose. This method employed the DELTA Taq DNApolymerase protocol developed in the present inventor's laboratory(Ranu, R. S., (1995), Biotechniques 18:390-395) or Thermo Sequenase®(Amersham, Inc.). Based on the analysis of the DNA sequence results, theACC synthase cDNA clones were identified as only one gene. FIG. 3 showsthe various landmarks, including start codon and termination signal. Thededuced amino acid sequence is shown in FIGS. 4.

Several additional features of these clones and several related cloneswhich include some noteworthy areas are described below. The cloneinclude two stop codons on the 5′ end of the untranslated region atpositions 169 and 178. The regular start codon is at position 271 andthe regular stop codon is at position 1711.

Development of Antibody Probes

Antibody probes were prepared for screening a cDNA expression libraryand for subsequent detection of ACC synthase gene products from plantcell extracts and for protein expressed from the cloned ACC synthaseDNA. Based on the sequence alignment data from tomato, three peptideswith largest stretches of amino acid sequence homology were selected.

(1) Peptide #1075, derived from the carboxy-terminus contained 35 aminoacid residues as follows:

NVSPGSSFLCSEPGWFRVCFANMDNATLDVALNRI   [SEQ ID NO: 7]

(2) Peptide #1076, derived from the amino terminus contained 33 aminoacids as follows:

YFDGWKAYDRDPYHSTKNSNGVIQMGLAENQLC   [SEQ ID NO: 8]

(3) Peptide #1077, from the middle region contained 38 amino acidresidues as follows:

YSLSKDMGMPGFRVGIIYSYNDRVVSTARRMSSFGLVS   [SEQ ID NO: 9]

These peptides were used to immunize rabbits. A 1:1 emulsion of 200μg/ml of peptide in complete Freund's Adjuvant was prepared, and 0.1 mlvolumes were injected subcutaneously (sc) into three different rabbitsat 17 to 18 sites on the animals' backs. Before injection, a preimmuneserum sample was obtained. On day 19 after the first immunization,rabbits received two intramuscular (im) injections of 0.35 ml of a 1:1emulsion of each peptide in incomplete Freund's adjuvant at 100 μg/ml.On day 35 after the first immunization, the day 19 im protocol wasrepeated. On day 92, each rabbit received a booster injection (im) withthe same peptide emulsion as on day 19. Seven days later, the rabbitswere bled, and serum was prepared.

Western blot analysis of antisera with the three peptides showed thepresence of antibodies against each of the three peptides and strongsignals indicating immunization was successful. Preimmune sera werenegative.

Expression of Cloned ACC Synthase Genes In Vitro

Use was made of the ZAP Express Vector system which contains abacteriophage T₃ promoter. Cloned ACC synthase genes are inserted byunidirectional EcoRI/XhoI site. The cloned insert can be excised fromthe phage in the form of a kanamycin-resistant pBK-CMV phagemid. Thedigestion of the phagemid from the three ACC synthase clones describedabove with NotI and BamI restriction enzymes showed the absence of theserestriction sites in the inserts.

DNA from clone pRoseKacc7 was prepared, linearized with NotI and usedfor in vitro transcription. The reaction mixture (100 μl) containedTris-HCl (pH7.9), 40 mM; MgCl₂, 6 mM; DTT, 10 mM; spermidine, 2 mM;m⁷GpppG, 1 mM; ATP, CTP and UTP, 0.5 mM each; GTP, 25 μM; Rnasin® (RNaseinhibitor), 120 units; DNA template, 1-2 μg; and T₃ RNA polymerase, 50units, as described in the inventor's publications. Samples wereincubated at 37° C. for 20 minutes. The GTP concentration in reactionmixture was raised to 0.5 mM, and incubation was continued for one hour.Aliquots (3-5 μl) of each reaction mixture were withdrawn and subjectedto agarose gel (1.2%) electrophoresis to determine the quality andefficiency of transcript synthesis. The analysis of transcript showedexpected size of RNA from each clone.

The in vitro transcripts from each clone were then translated at highefficiency using rabbit reticulocyte lysates as described by the presentinventor (Ranu, R. S., et al, 1979, Meth. Enzymol. 60:459-484) exceptthat they were made mRNA-dependent by treatment with micrococcalribonuclease. The in vitro translation products were immunoprecipitatedwith each of the three antisera described above or with a mixture of theantisera. The method used for immunoprecipitation and detection of ACCsynthase protein was by Western blotting as described by the presentinventor and colleagues in 1989 and recently published (Ranu, R. S., etal, (1996) Gene Expression 5:143-153). Translation products detectedfrom each cloned ACC synthase gene was the size expected based on thesize of the ORF of each clone. The in vitro translation productcomigrate with the in vivo product upon gel electrophoresis.

Regeneration and Transformation

For the geranium, petioles from very young immature leaves from activelygrowing plants of Pelargonium hortorum cv Samba (sincerity could alsohave been used) were harvested and sterilized in 15% clorox for 15minutes. They were then thoroughly rinsed with sterile distilled water(four times). The petioles were cut into 4-5 mm segments and cultured onmodified MS medium as further explained in A Revised Medium For RapidGrowth And Bioassys With Tabacco Tissue Cultures, Murashige T. andSkoog, F., Physiologia Plantarum 15,473-497 (1962). Modificationsconsist of one half concentration of major salts and pyridoxine HCl, 1mg/liter; nicotinic acid 1 mg/liter; and thiamine HCl 10 mg/liter. Inaddition, the medium used contained 5 μM BAP and 1 μM 1AA. Afterincubation of explants at 25° C. in the dark for three days, they weretransferred to light conditions. Regeneration became apparent by 15 daysand continued for five weeks. The small shoots are then subculturedindividually on MS medium containing 0.44 μM BAP and 0.11 μM IAA plus400 mg/liter of L-glutamine. After about five weeks, they developedfurther to about 3-4 cm long with 4-5 nodes. They were then subculturedon basal MS medium for rooting.

For transformation of geranium, pPHSacc41 was cut with Not I; thestaggered ends were filled-in with dGTP and dCTP using Kelnow DNApolymerase The other end was cut with Bam HI for ligation into anagrobacterium binary vector in reverse orientation. The vector wasprepared for ligation using HPA I and BamI. The ligated vector (withPHSacc41 in reverse orientation) was used to transform Agrobacteriumtumefaciens 2760.

The petiole explants were cocultivated with agrobacterium for 5-10minutes. After several days of cocultivation, agrobacterium cells werekilled in the presence of cefotaxime (400 μg/ml) and Kananeycin (200μg/ml). After about two weeks selection for transformants was continued.Ultimately, the transformed plants will be grown and tested for theirvarious properties to determine which had successfully achieved thedesired ACC synthase modification. The selected genetically alteredplants will be used to produce a new variety or line of plants whereinthe alteration is stably transmitted from generation to generation. Itis anticipated that the above regeneration and transformationprocedures—with some modifications as those of ordinary skill in the artwould readily understand—should work for the rose application as well.

The references cited above are all incorporated by reference herein,whether specifically stated as incorporated or not. Specifically, anyreferences mentioned in the application for this patent as well as allreferences listed in any information disclosure originally filed withthis or the priority application are hereby incorporated by reference intheir entirety to the extent such may be deemed essential to support theenablement of the invention(s), however, applicant disclaims making orsupporting any statements in said references which might be consideredinconsistent with the patentability of the following claims or anyaspect of the invention described.

Having now fully described this invention, it will be appreciated bythose skilled in the art that the same can be performed within a widerange of equivalent parameters, concentrations, and conditions withoutdeparting from the spirit and scope of the invention and without undueexperimentation. While this invention has been described in connectionwith specific embodiments thereof, it will be understood that it iscapable of further modifications. This patent covers any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth as follows in the scope of theappended claims. Further, it should be understood that variouspermutations and combination of the elements shown in the claims(whether method or apparatus) are possible and do fall within the scopeof this disclosure.

Deposits

The following illustrative plasmid encoding rose ACC synthase has beendeposited at the American Type Culture Collection, Rockville, Md. underthe requirements of the Budapest Treaty. This deposit has been grantedthe following accession number and is hereby incorporated by referenceto the extent permissible:

1. pRoseKacc7 cDNA clone comprising SEQ ID NO: 1—accession number ATCC98555.

9 1 1743 DNA Rosa kardinal 1 gccttggctt tcctcccttc gctttcttct tcttcttcttcatcatcgta ctctccgacg 60 acccgaaacc ccaccgcgac ccggcccgga tgtctccaatatgacccgga cccgagacga 120 agaccggcga cccagcagca gcagcagcgg cggcggaggaggcgccgatg agagttatag 180 tccctctaca aggcgtggtt caaggcagag gaggactcgttctcggctcc gtcataccat 240 gcgcgctctt ctatttcctc cagctttatc atgaaacgtcaccgttccaa ctccaacccg 300 ccgactccgc cgccttctcc ggactcggac tcggaccaccaccccgccgg gcagttggtg 360 gaagttccgg ttctgccccg gtcgatgtcg aggtcccatctctctccgag gaacccgggt 420 ccggtacatg tctcgggtcg ggccaattcg gttttgaaaggcggtgagcc gccgtattat 480 gtcggcttga ggaaggtggc ggaggatccg tacgacgagttgggtaaccc ggatggggtt 540 attcagctgg gtttggatga aaacaagtta gctttggacttggttcgaga ttggctactg 600 gagaatgcaa aggatgcaat actgggtggt gaggagcttgggattagtgg gattgcttgt 660 taccagcctt ctgatggttt aatggagctc aaactggctgtggcaggatt catgtctaag 720 gccatcggaa attcagttac gtacaacccc tcacaaattgtattgacagc tggtgcaacc 780 cctgcaattg agattctaag cttctgccta gcagacagtggaaacgcatt tctcgttccg 840 gcaccatatt accctggttt ggacagagat gtgaagtggcgaactggagt ggagataata 900 cctgttccat gccgcagtgc tgacaaattc aatttaagtataactgcact tgatcgagca 960 ttcaaccagg caaagaaacg tggtgtaaaa gttcgtgggattataatttc aaatccttca 1020 aatcctggtg gcagtttact tactcgtgaa tcactttacaaccttctgga ctttgcccga 1080 gagaagaaca ttcatataat ctcaaatgaa ttgtttgctggatccacgta tggaagtgaa 1140 gagtttgtta gcatggcaga aatcgttgat ttggaagatctcgaccagaa cagagtgcat 1200 atagtatatg gcatatcgaa agatctctca cttccaggtttcagggtggg tgccatctac 1260 tcctttaaca agaatgtctt gactgctgct aaaaagttgacaaggttctc ttctatctcc 1320 gccccatccc aacggttgct tatctctatg ctttcagacaccaaatttat gcataagttc 1380 atcgagatta acagagaaag gctccgtgga atgtatcttagatttgtgac aggattgaag 1440 caattgggca ttgagtgcac aaagagcaat gggggtttctactgttgggc agacttgagt 1500 gggttaattc gctcttacag tgagaaaggg gagcttgagctctgggatag gttgttgaat 1560 gtaggtaagc tcaatgttac tcctggatct tcttgtcattgtattgaacc gggatggttc 1620 cggttttgtt ttacgacgtt gactgaaaaa gatatccctgttgttataga acgaattcgg 1680 aatattgccg aaacatgtaa atcacacagt tgaaatgttcgttcattcta ctcaaaaaaa 1740 aa 1743 2 480 PRT Rosa kardinal 2 Met Lys ArgHis Arg Ser Asn Ser Asn Pro Pro Thr Pro Pro Pro Ser 1 5 10 15 Pro AspSer Asp Ser Asp His His Pro Ala Gly Gln Leu Val Glu Val 20 25 30 Pro ValLeu Pro Arg Ser Met Ser Arg Ser His Leu Ser Pro Arg Asn 35 40 45 Pro GlyPro Val His Val Ser Gly Arg Ala Asn Ser Val Leu Lys Gly 50 55 60 Gly GluPro Pro Tyr Tyr Val Gly Leu Arg Lys Val Ala Glu Asp Pro 65 70 75 80 TyrAsp Glu Leu Gly Asn Pro Asp Gly Val Ile Gln Leu Gly Leu Asp 85 90 95 GluAsn Lys Leu Ala Leu Asp Leu Val Arg Asp Trp Leu Leu Glu Asn 100 105 110Ala Lys Asp Ala Ile Leu Gly Gly Glu Glu Leu Gly Ile Ser Gly Ile 115 120125 Ala Cys Tyr Gln Pro Ser Asp Gly Leu Met Glu Leu Lys Leu Ala Val 130135 140 Ala Gly Phe Met Ser Lys Ala Ile Gly Asn Ser Val Thr Tyr Asn Pro145 150 155 160 Ser Gln Ile Val Leu Thr Ala Gly Ala Thr Pro Ala Ile GluIle Leu 165 170 175 Ser Phe Cys Leu Ala Asp Ser Gly Asn Ala Phe Leu ValPro Ala Pro 180 185 190 Tyr Tyr Pro Gly Leu Asp Arg Asp Val Lys Trp ArgThr Gly Val Glu 195 200 205 Ile Ile Pro Val Pro Cys Arg Ser Ala Asp LysPhe Asn Leu Ser Ile 210 215 220 Thr Ala Leu Asp Arg Ala Phe Asn Gln AlaLys Lys Arg Gly Val Lys 225 230 235 240 Val Arg Gly Ile Ile Ile Ser AsnPro Ser Asn Pro Gly Gly Ser Leu 245 250 255 Leu Thr Arg Glu Ser Leu TyrAsn Leu Leu Asp Phe Ala Arg Glu Lys 260 265 270 Asn Ile His Ile Ile SerAsn Glu Leu Phe Ala Gly Ser Thr Tyr Gly 275 280 285 Ser Glu Glu Phe ValSer Met Ala Glu Ile Val Asp Leu Glu Asp Leu 290 295 300 Asp Gln Asn ArgVal His Ile Val Tyr Gly Ile Ser Lys Asp Leu Ser 305 310 315 320 Leu ProGly Phe Arg Val Gly Ala Ile Tyr Ser Phe Asn Lys Asn Val 325 330 335 LeuThr Ala Ala Lys Lys Leu Thr Arg Phe Ser Ser Ile Ser Ala Pro 340 345 350Ser Gln Arg Leu Leu Ile Ser Met Leu Ser Asp Thr Lys Phe Met His 355 360365 Lys Phe Ile Glu Ile Asn Arg Glu Arg Leu Arg Gly Met Tyr Leu Arg 370375 380 Phe Val Thr Gly Leu Lys Gln Leu Gly Ile Glu Cys Thr Lys Ser Asn385 390 395 400 Gly Gly Phe Tyr Cys Trp Ala Asp Leu Ser Gly Leu Ile ArgSer Tyr 405 410 415 Ser Glu Lys Gly Glu Leu Glu Leu Trp Asp Arg Leu LeuAsn Val Gly 420 425 430 Lys Leu Asn Val Thr Pro Gly Ser Ser Cys His CysIle Glu Pro Gly 435 440 445 Trp Phe Arg Phe Cys Phe Thr Thr Leu Thr GluLys Asp Ile Pro Val 450 455 460 Val Ile Glu Arg Ile Arg Asn Ile Ala GluThr Cys Lys Ser His Ser 465 470 475 480 3 50 DNA Artificial Sequence Anoligonucleotide primer with XhoI restriction site 3 gagagagagagagagagaga actagtctcg agtttttttt tttttttttt 50 4 12 DNA ArtificialSequence EcoRI adaptor ligated to the blunt ends of cDNA 4 aattcggcag ag12 5 23 DNA Artificial Sequence i 3, 6, 9, 12, 18, and 21 PCR primer 5ggnytnccng gnttymgnrt ngg 23 6 23 DNA Artificial Sequence i 3, 5, 6, 15,and 18 PCR primer 6 canannckra asmanccnrs ytc 23 7 35 PRT Rosa kardinal7 Asn Val Ser Pro Gly Ser Ser Phe Leu Cys Ser Glu Pro Gly Trp 1 5 10 15Phe Arg Val Cys Phe Ala Asn Met Asp Asn Ala Thr Leu Asp Val 20 25 30 AlaLeu Asn Arg Ile 35 8 33 PRT Rosa kardinal 8 Tyr Phe Asp Gly Trp Lys AlaTyr Asp Arg Asp Pro Tyr His Ser Thr 1 5 10 15 Lys Asn Ser Asn Gly ValIle Gln Met Gly Leu Ala Glu Asn Gln Leu 20 25 30 Cys 9 38 PRT Rosakardinal 9 Tyr Ser Leu Ser Lys Asp Met Gly Met Pro Gly Phe Arg Val GlyIle 1 5 10 15 Ile Tyr Ser Tyr Asn Asp Arg Val Val Ser Thr Ala Arg ArgMet Ser 20 25 30 Ser Phe Gly Leu Val Ser 35

What is claimed is:
 1. An isolated DNA molecule consisting of SEQ IDNO:
 1. 2. A vector useful when introduced into a rose plant cell,comprising a polynucleotide hang the nucleotide sequence of SEQ ID NO:1, wherein the polynucleotide is inserted into the vector in reverseorientation.
 3. The vector according to claim 2 which expresses an RNAhaving a sequence complementary to an RNA having a sequence expressed bythe nucleotide sequence of SEQ ID NO:
 1. 4. The vector according toclaim 2 or 3 further comprising regulatory sequences required forexpression of the polynucleotide.
 5. The vector according to claim 4wherein said regulatory sequences comprise a promoter active in saidcell.
 6. The vector according to claim 5, wherein said regulatorysequences further comprise a polyadenylation signal.
 7. The vectoraccording to claim 5, wherein said promoter is a heterologous promoter.8. The vector according to claim 7, wherein said heterologous promoteris a viral promoter.
 9. The vector according to claim 8, wherein saidviral promoter is the CaMV 35S promoter.
 10. The vector according toclaim 7, wherein said heterologous promoter is selected from the groupconsisting of the SSU gene promoter, ribulose bisphosphate carboxylasepromoter, chlorophyll a/b binding protein promoter, potato ST-LS1 genepromoter, soybean heat shock protein hsp17.5-E promoter, soybean heatshock protein hsp17.3-B promoter, phenylalanine ammonia-lyase promoter,petunia 5-enolpyruvylshikimate-3-phosphate synthase gene promoter,Rhizobium meliloti FIXD gene promoter and nopaline synthase promoter.11. A rose cell transformed with the vector according to claim 2 or 3.12. A mature rose plant regenerated from a cell transformed with thevector according to claim 2 or
 3. 13. A transgenic part of a rose plantaccording to claim 12.